Screening of wine-indigenous strains reveals different responses to octanoic and decanoic acid stress.
In an attempt to evaluate the variability of the resistance of wine strains to MCFA, we compared the sensitivities of 76 indigenous strains to these inhibitors in a drop test (Table ). The strains were gathered in groups of similar sensitivities (see Table S1 in the supplemental material). Figure shows that wine strains present a high variability in their ability to resist MCFA. Resistance to decanoic acid generally is associated with a medium to strong resistance to octanoic acid. In contrast, four of the strains showing the highest resistance to octanoic acid were sensitive to decanoic acid. These results suggest strongly that wine yeast activate two partially overlapping mechanisms to resist these MCFA, and that some mechanisms involved in decanoic acid resistance may contribute to octanoic acid resistance.
FIG. 1. Variability of Saccharomyces cerevisiae wine strain resistance to octanoic acid (x axis) and decanoic acid (y axis) as revealed by drop test (resistance levels are given in Table ). The dimension of the spots is related to the number (more ...) Transcriptome analysis reveals that octanoic and decanoic acids activate two partially overlapping sets of genes.
To get further insights into the genes involved in the S. cerevisiae response to octanoic and decanoic acids, we studied the transcriptome of wine yeast strain U13, chosen for its high resistance to both inhibitors in the former experiment. This strain was exposed for 20 min to 50 μM each acid. For both acids these conditions were found to be sufficient for the induction of high resistance in a preliminary experiment.
We carried out competitive hybridizations in a triangular design between cDNA obtained from nonexposed cells (T) and cDNA obtained from octanoic acid (C8)- or decanoic acid (C10)-treated cells, as well as between cDNAs from the two acid-exposed conditions. Considering all the genes whose expression was significantly altered between the two conditions, we found that exposure to octanoic or decanoic acid affected 81 and 620 genes, respectively, compared to control cells, with 76 being common to both responses. The comparative hybridization of C8 and C10 modalities revealed that 71 genes were differently affected by these organic acids. The imbalance between the numbers of genes involved in each response suggested that cells were exposed to different stress intensities. To determine if the two responses were correlated, we selected the genes induced by decanoic acid (i.e., genes with significant C10/T log ratios) but that were not detected in the C8/C10 comparison. The C10/T log ratio of these genes was plotted against the C8/T log ratio of the same genes (Fig. ). The two ratios were highly correlated (R2 = 0.89), indicating that the two acids similarly affected this set of genes, and the slope of 0.6 reveals that the C8 response was weaker than the C10 response at the tested concentrations.
FIG. 2. Correlation between the two responses. Triangles, expression ratio of genes whose expression level varied significantly compared to that of the control for one modality; spheres, expression ratio of genes whose expression level varied significantly for (more ...)
To minimize the biological noise, we restricted the analysis to the genes whose expression significantly differed by a minimum log ratio of 0.5 for C10 acid compared to that of the control. A ratio of 0.3 was chosen for C8 acid compared to that of the control to obtain similar cutoffs for the two acids. As a consequence, compared to the control, 75 genes were significantly modulated by octanoic acid and 165 by decanoic acid, with 53 genes being shared by the two responses (Fig. ). The analysis of the functional categories through gene ontology (MIPS) (Table ) shows that the yeast responses to these two organic acids share almost the same subset of genes involved in cell energy supply. Decanoic acid response also includes sets of genes involved in ribosome biogenesis and RNA processing. In agreement with this, GO biological processes for decanoic acid response include ribosome and large ribosomal subunit biogenesis (GO:0042254 and GO:0042273) and oxidation reduction (GO:0055114). The m
uperfamily proteins (MFS) also are especially well represented in this response. In addition, the analysis of these responses with Eu.Gene 1.2.1 (10
) showed the significant activation of the fatty acid beta oxidation pathway.
FIG. 3. Venn diagram presenting the genes differentially expressed in these three conditions. C8, 20-min exposure to octanoic acid at 50 mM; C10, 20-min exposure to octanoic acid at 50 mM; C8/C10, comparison of exposure to C10 to exposure to C8. Repressed genes (more ...)
Classification of genes involved in response to octanoic and decanoic acid according to MIPS functional categories
Direct comparison of octanoic and decanoic acid-treated cells (with a log ratio of 0.3) allowed us to gain further indications on the analogies/differences between the two responses. We found that 68 genes presented significantly different expression profiles (Fig. ). As the activation of genes was not similar for octanoic acid and decanoic acid, we divided the activated genes in three sets: genes activated by both C8 and C10 but not by the control were qualified as shared responses, the genes activated by C8 but not the control or C10 were called C8-specific responses, and the genes activated by C10 but not the control or C8 combined with genes differentially expressed after exposure to C10 were called C10-specific responses (Fig. ).
Among the 53 genes whose expression was affected by both acids (C8 and C10 shared response) (see Table S2 in the supplemental material), 39 were upregulated and 14 repressed. Among them, C8 more efficiently induced PDR12 (3.5 times increase), whereas C10 more specifically induced ALD4, CWP1, TMA17, and HXK1.
The octanoic acid-specific response included 22 genes; 8 were upregulated (i.e., IDH2, ATP3, ALG12, TRX2, etc.), whereas 14 were repressed (EFT1, EFT2, ZRT1, FAS1, etc.).
The exposure to C10 resulted in the specific modulation of the expression of 114 genes (C10-specific response), among which EEB1, coding for an ethyl ester synthase, tops the list for its high induction (8.4 times increase). Two other genes (FAA1 and ELO1) involved in fatty acid metabolism also were induced (1.7 and 2.4 times increase), suggesting a potential metabolism of the fatty acid. However, two transporters involved in cell detoxification, TPO4 and PDR12 (two times higher expression), and, to a lesser extent, TPO1 (1.5 times increase), also were activated.
Comparison of octanoic and decanoic acid responses to stress responses already described.
The octanoic and decanoic acid responses were compared to those already described for different stresses (Table ) after a similar incubation period: sorbic acid (a weak acid response has been described [36
]), sodium dodecyl sulfate (39
), octanol (14
), fluphenazine (12
), benomyl (27
), 2,4-deoxyphenoxyacetic acid (44
), polyoxyethylene-9-laurylether (POELE), 2,4-dichlorophenol (DCP) (37
), oleic acid oxidative stress (23
) and H2
). For each pair of stresses, we have counted the number of genes significantly induced or repressed by both stresses. When the significance information was not available, we selected genes induced (or repressed) by at least a factor of 2. The highest similarities were observed between the responses to octanoic and decanoic acid (71% of C8 responses are shared with C10 responses), but about half of the genes involved in both responses also are shared with H2
oxidative stress. Significant portions of these responses also are activated by detergent stresses: i.e., 45 and 35% of genes activated by octanoic and decanoic acids also are activated by SDS. Octanoic acid response presents 29% of genes in common with the sorbic acid response. In contrast with these different stresses, the oleic acid oxidative stress involved few of the genes activated by C8 or C10.
Comparison of different transcriptional responsesa
As a consequence, our results suggest that the responses to octanoic and decanoic acid are composite responses involving the organic weak acid response and a detergent response, with both of them presenting many similarities with an oxidative stress.
Search for known transcription factors involved in MCFA response.
For both acid responses as well as for the other stresses cited in the former paragraph, we obtained from the Yeastract web site (43
) the transcription factors involved in the regulation of each gene and scored the number of genes regulated per transcription factor. These scores were compared in a correspondence analysis. The result plotted in Fig. revealed that the octanoic acid response presents, as expected, similarities to the weak acid response involving War1 and Msn4 (Fig. ). In contrast, the C10 response appeared much closer to the SDS stress response. C8 and C10 responses also appeared as potentially regulated by transcription factors HAP1
, and HAP5
. This could be interpreted as a sign of the activation of the fatty acid beta-oxidation pathway.
FIG. 4. Factorial component analysis comparing the involvement of each transcription factor in the different stress responses. The proportion of each stress response explained by one transcription factor has been calculated from the Yeastract website. Codes for (more ...) Phenotyping screening of Euroscarf deletion mutant strains allows us to identify genes involved in resistance. (i) Transporters.
Looking for transporters involved in octanoic and decanoic acid expulsion, we screened a collection of haploid strains deleted for their PDR genes as well as for other transporters, including ADP1, AQR1, ATM1, AUS1, AZR1, BPT1, DIP5, FLR1, NFT1, PDR5, PDR10, PDR11, PDR12, PDR15, PDR18, PXA2, SNQ2, TPO1, TPO2, TPO3, TPO4, YBT1, and YOR1. Figure shows the results obtained for the sensitivity test of some of these strains in the presence of 0 to 0.6 mM C8 and 0 to 0.25 mM C10.
FIG. 5. Drop test presenting the sensitivity provoked by the deletion of different transporters on YPD (pH 4.5) medium containing 0.6 mM octanoic acid (C8) or 0.25 mM decanoic acid (C10) compared to that of the control (T). Cells used to prepare the spots were (more ...)
The highest sensitivity to octanoic acid was obtained for the Δpdr12 strain; however, the deletion of TPO1 also resulted in an increased sensitivity to this inhibitor. In addition, the Δpdr12-Δtpo1 double deletant strain was found to be more sensitive than the two single-deletion strains, showing a cumulative effect of the two transporters in the expulsion of this acid. In contrast to octanoic acid, the Δtpo1 strain exhibited the highest sensitivity to decanoic acid, while the deletion of PDR12 did not affect this phenotype. However, Δtpo4 and Δsnq2 strains also were slightly affected, indicating a possible ability of these transporters to expulse decanoic acid. None of the other strains tested showed modified resistance to octanoic or decanoid acid. Since Pdr12p and Tpo1p were the main transporters of octanoic and/or decanoic acid, we constructed diploid strains harboring one or two deleted copies of the genes (Fig. ). In a background where two alleles of a given transporter were present, the presence of a single allele of the other transporter was sufficient to regenerate the wild-type phenotype. When the two tested genes were present as a single copy, the growth of yeast strains was injured on both octanoic and decanoic acids. Moreover, when a single copy of the TPO1 gene was present, decanoic acid resistance was correlated with the number of copies of the PDR12 gene, indicating that Pdr12p plays a part, though discrete, in resistance to decanoic acid. This observation was confirmed by analyzing the growth of the diploid on liquid medium complemented with inhibitors (Bioscreen analysis) (results not shown).
FIG. 6. Drop test presenting the sensitivity of diploid strains deleted from one or two copies of PDR12 and TPO1 transporter genes on YPD (pH 4.5) medium containing 0.6 mM octanoic acid (C8) or 0.25 mM decanoic acid (C10) compared to that of the control (T). (more ...) (ii) Transcription factors.
We also tested Euroscarf strains deleted for regulatory genes, including main regulators of stress responses (PDR1/PDR3, MSN2/MSN4, HSP30, etc.), transcription factors already described in weak organic acid stress or in acid stress (WAR1, HAA1, etc.), and other transcription factors suggested by the transcriptome analysis. Tested transcription factors were ADR1, AFT1, ARR1, CIN5, CRZ1, KFH2, FLO8, GCN4, GCR2, HAA1, HAC1, HAP2 to HAP5, HSF1, HSP30, MSN2, MSN4, NRG1, OAF1, PDR1, PDR3, PIP2, RPN4, SEF1, SKO1, SOK2, STB5, TOS8, WAR1, YAP1, YAP2, and YRR1. Figure shows the results obtained for the drug sensitivity test of some of these strains in the presence of 0 to 0.6 mM C8 and 0 to 0.25 mM C10.
FIG. 7. Drop test presenting the sensitivity of strains deleted for different transcription factors on YPD (pH 4.5) medium containing 0.6 mM octanoic acid (C8) or 0.25 mM decanoic acid (C10) compared to that of the control (T). Cells used to prepare the spots (more ...)
The octanoic acid response is clearly under the control of the WAR1
transcription factor. The deletion of PDR3
also has a great impact on this phenotype, while PDR1
deletion does not modify strain resistance to octanoic acid. None of the other transcription factors that were tested seemed to be involved in the modulation of the octanoic acid response, including HAA1
(not shown), which was described to be involved in a weak acid response (13
The response to decanoic acid is clearly under the control of PDR1
, while PDR3
deletion does not result in higher sensitivity. However, the Δpdr1
double mutant is more sensitive to decanoic acid than the Δpdr1
mutant, indicating a slight role of PDR3
. Moreover, the deletion of STB5
also lowers strain resistance to decanoic acid. This transcription factor is known to form a heterodimer with Pdr1p but not with Pdr3p and to regulate the pentose phosphate pathway and NADPH production in response to oxidative stress (25
). Three other transcription factors are clearly involved in response to decanoic acid, namely, NRG1
, and PIP2
Finally, the deletion of MSN2 (but not MSN4) or YAP1 seems to have little effect on decanoic acid resistance.
(iii) Other genes.
We also tested some other genes already described to be involved in weak acid resistance or in lipophilic compound resistance. We observed that PDR16 deletion had an impact on octanoic acid resistance, while the deletion of the homologous PDR17 gene had no impact.
Finally, SOD1 deletion had a drastic effect on both octanoic and decanoic acid sensitivity, while SOD2 deletion had no impact on these phenotypes.